Field of the Invention
This invention relates to an apparatus for real-time detecting a polynucleotide product obtained from a polymerase chain reaction (PCR).
Description of the Related Art
A PCR is a cyclic enzyme reaction to replicate a DNA chain. As the PCR is used as a template of a cycle in which PCR products (nucleic acid amplification products) replicated in previous cycles are consecutive, arrayed target molecules can be exponentially amplified. A real time PCR is to excite fluorescent material by irradiating a PCR product with excitation light using, for example, array-specific probes (TAQMAN probes) marked with two different kinds of fluorescent pigments interfering with each other, measure the strength of the fluorescence and monitor amplification of the PCR products in real time.
In quantitative use, a threshold ((6) in FIG. 7) is set in an exponential amplification region of an amplification curve for existing samples, and a point (threshold cycle number (Ct). (8) in FIG. 8) at which the threshold intersects the amplification curve is calculated. There is a linear relation between the threshold cycle number (Ct) and the initial amount of DNA of a test sample measured in terms of log value, and a calibration curve representing this linear relation can be prepared. The initial amount of DNA of the test sample is estimated based on the calibration curve. This enables correct quantitativeness based on a PCR amplification speed theory.
Here, since an actual PCR efficiency is not 100%, the concentration of an amplified PCR product is expressed by the following Equation 1.[DNA]=[DNA]0(1+e)c  (1)
Where, [DNA]: Concentration of PCR product                [DNA]0: Initial concentration of target Template                    e: Average PCR efficiency            c: Cycle number                        
That is, if the average PCR efficiency (e) is 100% (i.e., e=1 in the above Equation (1)), although the concentration [DNA] of the PCR product is exponentially amplified with 2c, since the efficiency (e) is slowly lowered from the initial stage, through the middle stage, to the late state of the cycle, an amplification curve is as shown in FIG. 7. In FIG. 7, a horizontal axis represents the cycle number and a vertical axis represents the fluorescence strength. As shown in the figure, the fluorescence strength is exponentially amplified ((5) in FIG. 7) at the cycle initial stage, linearly amplified ((6) in FIG. 7) at the cycle middle stage, and not amplified ((7) in FIG. 7) by a plateau effect at the cycle late stage.
Chemical-reactive factors for this plateau effect are as follows.                Hydrolysis of dNTP and primer        Deactivation of DNA polymerase (DNA synthase to make a copy of a template (cast)) by heat.        Lowering of primer annealing efficiency by re-association of one chain PCR fragment        Competitive material by non-specific PCR product        Accumulation of PCR inhabitation material such as pyrophosphate        Hydrolysis of PCR product by exonuclease activation of DNA polymerase        
Accordingly, the measurement in the exponential amplification region satisfying the relation of the Equation (1) is a precondition for the real time PCR (see Patent Document 1)
[Patent Document 1] Japanese Patent Application Publication No. 2005-516630
[Patent Document 2] Japanese Patent No. 2909216
As a reactive vessel used for the real time PCR, a vessel called a micro plate having a plurality (for example, 96) of wells (reactive regions constituted by concave portions) is being used in common and reactive solution having a predetermined initial DNA concentration is divisionally poured in the wells. However, an amplification curve for each of the wells of the reactive vessel becomes unbalanced due to the following apparatus error factors                Error of optical system        Concentration error of correction solution        Divisional pour error of correction solution        Light transmission error of cap of reactive vessel or seal film        Contamination error of reactive vessel        Divisional pour error of reactive vessel        
Here, the reactive vessel mainly uses a cheap method in which the above-mentioned seal film with an adhesive is attached to the entire region of a single side and the wells are cover by a cap. In addition, the wells are irradiated with excitation light through the seal film and fluorescence generated from the PCR product (reaction product) is detected by a light detecting part such as a CCD camera through the seal film (these components constitute an optical system). In this manner, although the seal film and the body of the reactive vessel constitute important factors of the optical system in measurement of the fluorescence strength, these components are consumable parts, it is difficult to expect optical performance with high uniformity and precision.
FIG. 4 shows an actual image of a reactive vessel before PCR, which is detected by an optical detecting part. While the circumference of wells of the reactive vessel appears to be black as a whole, the brightness of pixels of the image as a background is not necessarily constant and there occurs a spot due to contamination of the optical system or way-out light as indicated by (1) in the figure. When a PCR reaction is initiated, this spot overlaps with images (96 images appearing to be round in FIG. 5) of the wells as indicated by (2) in FIG. 5, wastefully adding to the fluorescence strength of the wells.
So, in the prior art, empty reactive vessels containing no DNA are initially prepared, and fluorescence strengths for wells are measured in such an empty state and are stored as standard correction values. Then, by performing a correcting process in which the stored correction values are subtracted from measurement values of actual fluorescence strengths, such correction of the optical system is performed. However, since errors due to contamination of the empty reactive vessels are inherent to the respective reactive vessels, if correction values by other standard empty reactive vessels are used, there occurs a problem of errors in measurement values.
In addition, in Patent Document 2, although a first fluorescence signal is corrected with a second fluorescence signal, since a solution that generates second fluorescence for reference must be added to a solution that generates first fluorescence to be originally measured, work becomes complicated and costs are raised. In addition, the solution that generates the second fluorescence can not give any effect if it can not be divisionally poured and measured with very high precision. In addition, since an especial optical filter has to be used to measure the second fluorescence and has to be exchanged for the solution that generates the first fluorescence and the solution that generates the second fluorescence every measurement, there is a problem that it takes extra time to acquire and process data.
The present invention has made to overcome the above technical problems and it is an object of the invention to provide an apparatus for detecting a nucleic acid amplification product in real time, which is capable of effectively excluding or reducing apparatus error factors without using a second fluorescence signal used for correction.